The functional group of a protein that can react with the functional group of a dye molecule can be inferred from the amino acid of the protein. Specific examples include the amino group (—CH2CH2CH2CH2NH2) of lysine, the thiol group (—CH2SH) of cysteine, the imidazolamino group
of histidine, the secondary aliphatic hydroxyl group (—CH2CH(OH)CH3) of threonine, the primary aliphatic hydroxyl group (—CH2OH) of serine, the phenol hydroxyl group
of tyrosine, and so forth. The binding may also occur at the N-terminal amino group (—COCHRNH2) of an amino acid. Also, the functional group of a dye may bind to biomolecules such as sugars, glycoproteins, antibodies, and so forth.
The functional groups of the currently known dyes or other substances used to label biomolecules may be classified according to the functional groups of the biomolecules to which they bind to. They are frequently referred to by their common names.
The most frequently used functional groups for binding to the amino group of a protein molecule are succinimidyl ester and isothiocyanate, and the functional group the most frequently used for binding to the thiol group of a protein molecule is maleimide. For binding to the hydroxyl group of a protein molecule, the following functional groups are used:

In addition to the above-described functional groups, many researchers and companies are designing intermediates exhibiting better performance. Although they exhibit fast reaction with most biomolecules as well as superior binding ability, they are usually unstable in aqueous solutions or susceptible to heat and byproducts are often formed after the reaction as the leaving group is detached.
Recently, water-soluble fluorescent dyes are used actively in the bioindustries. For a water-soluble fluorescent dye to be introduced into biomolecules, it needs to experience less photobleaching and quenching in aqueous solutions or under hydrophilic conditions, have a large molar extinction coefficient so as to absorb a large amount of light, fluoresce in the visible or near-infrared region of 500 nm or above distant from the fluorescence range of the biomolecule itself, and be stable under various pH conditions. Due to these requirements, the structures of dyes that can be used for biomolecule labeling are restricted.
Not all dyes fluoresce. Researchers of various fields have developed dyes having chromophores capable of fluorescing. Representative examples of fluorophores known to date include anthranilates, 1-alkylthic isoindoles, pyrrolinones, bimanes, benzoxazoles, benzimidazoles, benzofurazans, naphthalenes, coumarins, stilbenes, carbazoles, phenanthridines, anthracenes, acridines, fluoresceins, eosins, rhodamines, pyrenes, chrysenes, and the like. Derivatives similar to those fluorophores in structure are also studied. These fluorophores are incorporated into various functional groups for binding to biomolecules and are commercially available as various products.
It is to be noted that these fluorescent dyes should exhibit strong fluorescence in media in which most biomolecules are present, that is, in aqueous solutions, in order that the dyes are applicable to the field of biology. The most commonly used fluorescent dyes for such application include xanthan-based fluoresceins and rhodamines and polymethine-based cyanines. For various applications, numerous researchers have introduced pigments and fluorescent dyes for labeling biomolecules into which functional groups capable of binding to protein nucleophiles, i.e. amino, thiol and hydroxyl group, and electrophiles, i.e. aldehyde, ketone and carboxylic acid groups, are incorporated.
Generally, cyanine dyes exhibit excellent optical and pH stability, have narrow absorption and emission wavelength ranges, and fluoresce in the range of 500-800 nm. Since this fluorescence range does not overlap with the autofluorescence range of biomolecules, it is easy to analyze. In addition, the cyanine dyes exhibit high molar extinction coefficients, although there are slight differences depending on solvents and solubility characteristics. The following chemical formulas are the general structures of cyanine dyes known in the literatures and the basic structures of hetero compounds known as derivatives.

The most commonly used cyanine dyes commercially available are the compounds having indoles as hetero rings and succinimidyl ester groups capable of binding to the amino group of an amino acid as reactive functional groups. For example, GE Healthcare's U.S. Pat. Nos. 5,268,486, 6,043,025 and 6,127,134 and WO96/33406 disclose introduction of various succinimidyl ester groups into cyanine dyes and labeling of biomolecules such as antibodies, peptides, etc. The following chemical formulas are representative structures of cyanine dyes, which are commercially available from GE Healthcare under the trade names Cy3, Cy5 and Cy7.

However, since the succinimidyl ester groups are unstable in aqueous solutions, the dyes cannot be maintained stably for a long reaction time. Also, there is a problem that N-hydroxysuccinimide is necessarily produced as a byproduct.